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My laboratory studies the strategies pathogens utilize to colonize and subvert the epithelial barrier. We have focused on the epithelial junctions as a target for bacterial pathogens, since the cell-cell junctions serve as both a barrier to infection and also a major control site for epithelial function. In particular, we are interested in how the gastric pathogen Helicobater pylori may cause cancer by interfering with cell signaling at the epithelial junctions. We are also studying how various bacteria cross and invade the epithelium. For example, we recently found that Listeria monocytogenes targets a specialized subset of cell-cell junctions at the tip of the intestinal villi to find its receptor for invasion. We are interested in determining whether this mode of gastrointestinal invasion of the epithelium is also used by other gastrointestinal pathogens.

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Abstract

Helicobacter pylori's ability to respond to environmental cues in the stomach is integral to its survival. By directly visualizing H. pylori swimming behavior when encountering a microscopic gradient consisting of the repellent acid and attractant urea, we found that H. pylori is able to simultaneously detect both signals, and its response depends on the magnitudes of the individual signals. By testing for the bacteria's response to a pure acid gradient, we discovered that the chemoreceptors TlpA and TlpD are each independent acid sensors. They enable H. pylori to respond to and escape from increases in hydrogen ion concentration near 100 nanomolar. TlpD also mediates attraction to basic pH, a response dampened by another chemoreceptor TlpB. H. pylori mutants lacking both TlpA and TlpD (ΔtlpAD) are unable to sense acid and are defective in establishing colonization in the murine stomach. However, blocking acid production in the stomach with omeprazole rescues ΔtlpAD's colonization defect. We used 3D confocal microscopy to determine how acid blockade affects the distribution of H. pylori in the stomach. We found that stomach acid controls not only the overall bacterial density, but also the microscopic distribution of bacteria that colonize the epithelium deep in the gastric glands. In omeprazole treated animals, bacterial abundance is increased in the antral glands, and gland colonization range is extended to the corpus. Our findings indicate that H. pylori has evolved at least two independent receptors capable of detecting acid gradients, allowing not only survival in the stomach, but also controlling the interaction of the bacteria with the epithelium.

Abstract

The constant regeneration of stomach epithelium is driven by long-lived stem cells, but the mechanism that regulates their turnover is not well understood. We have recently found that the gastric pathogen Helicobacter pylori can activate gastric stem cells and increase epithelial turnover, while Wnt signalling is known to be important for stem cell identity and epithelial regeneration in several tissues. Here we find that antral Wnt signalling, marked by the classic Wnt target gene Axin2, is limited to the base and lower isthmus of gastric glands, where the stem cells reside. Axin2 is expressed by Lgr5+ cells, as well as adjacent, highly proliferative Lgr5- cells that are able to repopulate entire glands, including the base, upon depletion of the Lgr5+ population. Expression of both Axin2 and Lgr5 requires stroma-derived R-spondin 3 produced by gastric myofibroblasts proximal to the stem cell compartment. Exogenous R-spondin administration expands and accelerates proliferation of Axin2+/Lgr5- but not Lgr5+ cells. Consistent with these observations, H. pylori infection increases stromal R-spondin 3 expression and expands the Axin2+ cell pool to cause hyperproliferation and gland hyperplasia. The ability of stromal niche cells to control and adapt epithelial stem cell dynamics constitutes a sophisticated mechanism that orchestrates epithelial regeneration and maintenance of tissue integrity.

Abstract

Colonization of the human stomach by Helicobacter pylori and its role in causing gastric cancer is one of the richest examples of complex relationship among human cells, microbes, and their environment. It is also a puzzle of enormous medical importance given the incidence and lethality of gastric cancer worldwide. We review recent findings that have changed how we view these relationships and affected the direction of gastric cancer research. For example, recent data indicate that subtle mismatches between host and microbe genetic traits greatly affect risk of gastric cancer. The ability of H pylori and its oncoprotein CagA to reprogram epithelial cells and activate properties of stemness demonstrates the sophisticated relationship among H pylori and progenitor cells in the gastric mucosa. The observation that cell-associated H pylori can colonize the gastric glands and directly affect precursor and stem cells supports these observations. The ability to mimic these interactions in human gastric organoid cultures as well as animal models will allow investigators to more fully unravel the extent of H pylori control on the renewing gastric epithelium. Finally, our realization that external environmental factors, such as dietary components and essential micronutrients, as well as the gastrointestinal microbiota, can change the balance between H pylori's activity as a commensal or a pathogen has provided direction to studies aimed at defining the full carcinogenic potential of this organism.

Abstract

Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

Abstract

The gastric pathogen Helicobacter pylori interacts intimately with the gastric mucosa to avoid the microbicidal acid in the stomach lumen. The cues H. pylori senses to locate and colonize the gastric epithelium have not been well defined. We show that metabolites emanating from human gastric organoids rapidly attract H. pylori. This response is largely controlled by the bacterial chemoreceptor TlpB, and the main attractant emanating from epithelia is urea. Our previous structural analyses show that TlpB binds urea with high affinity. Here we demonstrate that this tight binding controls highly sensitive responses, allowing detection of urea concentrations as low as 50 nM. Attraction to urea requires that H. pylori urease simultaneously destroys the signal. We propose that H. pylori has evolved a sensitive urea chemodetection and destruction system that allows the bacterium to dynamically and locally modify the host environment to locate the epithelium.

Abstract

Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology.We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology.H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5(+) stem cell proliferation, and up-regulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori.H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.

Abstract

Staphylococcus aureus is both a major bacterial pathogen as well as a common member of the human skin microbiota. Due to its widespread prevalence as an asymptomatic skin colonizer and its importance as a source of skin and soft tissue infections, an improved understanding of how S. aureus attaches to, grows within, and breaches the stratified layers of the epidermis is of critical importance. Three-dimensional organotypic human skin culture models are informative and tractable experimental systems for future investigations of the interactions between S. aureus and the multi-faceted skin tissue. We propose that S. aureus virulence factors, primarily appreciated for their role in pathogenesis of invasive infections, play alternative roles in promoting asymptomatic bacterial growth within the skin. Experimental manipulations of these cultures will provide insight into the many poorly understood molecular interactions occurring at the interface between S. aureus and stratified human skin tissue.

Abstract

Gastric adenocarcinoma is strongly associated with Helicobacter pylori infection; however, most infected persons never develop this malignancy. H. pylori strains harboring the cag pathogenicity island (cag+), which encodes CagA and a type IV secretion system (T4SS), induce more severe disease outcomes. H. pylori infection is also associated with iron deficiency, which similarly augments gastric cancer risk. To define the influence of iron deficiency on microbial virulence in gastric carcinogenesis, Mongolian gerbils were maintained on iron-depleted diets and infected with an oncogenic H. pylori cag+ strain. Iron depletion accelerated the development of H. pylori-induced premalignant and malignant lesions in a cagA-dependent manner. H. pylori strains harvested from iron-depleted gerbils or grown under iron-limiting conditions exhibited enhanced virulence and induction of inflammatory factors. Further, in a human population at high risk for gastric cancer, H. pylori strains isolated from patients with the lowest ferritin levels induced more robust proinflammatory responses compared with strains isolated from patients with the highest ferritin levels, irrespective of histologic status. These data demonstrate that iron deficiency enhances H. pylori virulence and represents a measurable biomarker to identify populations of infected persons at high risk for gastric cancer.

Abstract

Microbes use directed motility to colonize harsh and dynamic environments. We discovered that Helicobacter pylori strains establish bacterial colonies deep in the gastric glands and identified a novel protein, ChePep, necessary to colonize this niche. ChePep is preferentially localized to the flagellar pole. Although mutants lacking ChePep have normal flagellar ultrastructure and are motile, they have a slight defect in swarming ability. By tracking the movement of single bacteria, we found that ΔChePep mutants cannot control the rotation of their flagella and swim with abnormally frequent reversals. These mutants even sustain bursts of movement backwards with the flagella pulling the bacteria. Genetic analysis of the chemotaxis signaling pathway shows that ChePep regulates flagellar rotation through the chemotaxis system. By examining H. pylori within a microscopic pH gradient, we determined that ChePep is critical for regulating chemotactic behavior. The chePep gene is unique to the Epsilonproteobacteria but is found throughout this diverse group. We expressed ChePep from other members of the Epsilonproteobacteria, including the zoonotic pathogen Campylobacter jejuni and the deep sea hydrothermal vent inhabitant Caminibacter mediatlanticus, in H. pylori and found that ChePep is functionally conserved across this class. ChePep represents a new family of chemotaxis regulators unique to the Epsilonproteobacteria and illustrates the different strategies that microbes have evolved to control motility.Helicobacter pylori strains infect half of all humans worldwide and contribute to the development of peptic ulcers and gastric cancer. H. pylori cannot survive within the acidic lumen of the stomach and uses flagella to actively swim to and colonize the protective mucus and epithelium. The chemotaxis system allows H. pylori to navigate by regulating the rotation of its flagella. We identified a new protein, ChePep, which controls chemotaxis in H. pylori. ChePep mutants fail to colonize the gastric glands of mice and are completely outcompeted by normal H. pylori. Genes encoding ChePep are found only in the class Epsilonproteobacteria, which includes the human pathogen Campylobacter jejuni and environmental microbes like the deep-sea hydrothermal vent colonizer Caminibacter mediatlanticus, and we show that ChePep function is conserved in this class. Our study identifies a new colonization factor in H. pylori and also provides insight into the control and evolution of bacterial chemotaxis.

Abstract

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche.

Abstract

Listeria monocytogenes (Lm) uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ) that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF) increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine.

Abstract

Helicobacter pylori (Hp) intimately interacts with the gastric epithelial surface and translocates the virulence factor CagA into host cells in a contact-dependent manner. To study how Hp benefits from interacting with the cell surface, we developed live-cell microscopy methods to follow the fate of individual bacteria on the cell surface and find that Hp is able to replicate and form microcolonies directly over the intercellular junctions. On polarized epithelia, Hp is able to grow directly on the apical cell surface in conditions that do not support the growth of free-swimming bacteria. In contrast, mutants in CagA delivery are defective in colonization of the apical cell surface. Hp perturbs the polarized epithelium in a highly localized manner, since wild-type Hp does not rescue the growth defect of the CagA-deficient mutants upon co-infection. CagA's ability to disrupt host cell polarity is a key factor in enabling colonization of the apical cell surface by Hp, as disruption of the atypical protein kinase C/Par1b polarity pathway leads to rescue of the mutant growth defect during apical infection, and CagA-deficient mutants are able to colonize the polarized epithelium when given access to the basolateral cell surface. Our study establishes the cell surface as a replicative niche and the importance of CagA and its effects on host cell polarity for this purpose.

Abstract

Helicobacter pylori are spiral-shaped gram-negative bacteria with polar flagella that live near the surface of the human gastric mucosa. They have evolved intricate mechanisms to avoid the bactericidal acid in the gastric lumen and to survive near, to attach to, and to communicate with the human gastric epithelium and host immune system. This interaction sometimes results in severe gastric pathology. H pylori infection is the strongest known risk factor for the development of gastroduodenal ulcers, with infection being present in 60%-80% of gastric and 95% of duodenal ulcers.(1)H pylori is also the first bacterium to be classified as a definite carcinogen by the World Health Organization's International Agency for Research on Cancer because of its epidemiologic relationship to gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma.(2) In the last 25 years, since H pylori was first described and cultured, a complete paradigm shift has occurred in our clinical approach to these gastric diseases, and more than 20,000 scientific publications have appeared on the subject. From the medical point of view, H pylori is a formidable pathogen responsible for much morbidity and mortality worldwide. However, H pylori infection occurs in approximately half of the world population, with disease being an exception rather than the rule. Understanding how this organism interacts with its host is essential for formulating an intelligent strategy for dealing with its most important clinical consequences. This review offers an insight into H pylori host-bacterial interactions.

Abstract

Listeria monocytogenes causes invasive disease by crossing the intestinal epithelial barrier. This process depends on the interaction between the bacterial surface protein Internalin A and the host protein E-cadherin, located below the epithelial tight junctions at the lateral cell-to-cell contacts. We used polarized MDCK cells as a model epithelium to determine how L. monocytogenes breaches the tight junctions to gain access to this basolateral receptor protein. We determined that L. monocytogenes does not actively disrupt the tight junctions, but finds E-cadherin at a morphologically distinct subset of intercellular junctions. We identified these sites as naturally occurring regions where single senescent cells are expelled and detached from the epithelium by extrusion. The surrounding cells reorganize to form a multicellular junction that maintains epithelial continuity. We found that E-cadherin is transiently exposed to the lumenal surface at multicellular junctions during and after cell extrusion, and that L. monocytogenes takes advantage of junctional remodeling to adhere to and subsequently invade the epithelium. In intact epithelial monolayers, an anti-E-cadherin antibody specifically decorates multicellular junctions and blocks L. monocytogenes adhesion. Furthermore, an L. monocytogenes mutant in the Internalin A gene is completely deficient in attachment to the epithelial apical surface and is unable to invade. We hypothesized that L. monocytogenes utilizes analogous extrusion sites for epithelial invasion in vivo. By infecting rabbit ileal loops, we found that the junctions at the cell extrusion zone of villus tips are the specific target for L. monocytogenes adhesion and invasion. Thus, L. monocytogenes exploits the dynamic nature of epithelial renewal and junctional remodeling to breach the intestinal barrier.

Helicobacter pylori CagA induces a transition from polarized to invasive phenotypes in MDCK cellsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABagnoli, F., Buti, L., Tompkins, L., Covacci, A., Amieva, M. R.2005; 102 (45): 16339-16344

Abstract

CagA is a bacterial effector protein of Helicobacter pylori that is translocated via a type IV secretion system into gastric epithelial cells. We previously described that H. pylori require CagA to disrupt the organization and assembly of apical junctions in polarized epithelial cells. In this study, we provide evidence that CagA expression is not only sufficient to disrupt the apical junctions but also perturbs epithelial differentiation. CagA-expressing cells lose apicobasal polarity and cell-cell adhesion, extend migratory pseudopodia, and degrade basement membranes, acquiring an invasive phenotype. Expression of the CagA C-terminal domain, which contains the tyrosine phosphorylated EPIYA motifs, induces pseudopodial activity but is not sufficient to induce cell migration. Conversely, the N terminus targets CagA to the cell-cell junctions. Neither domain is sufficient to disrupt cell adhesion or cell polarity, but coexpressed in trans, the N terminus determines the localization of both polypeptides. We show that CagA induces a morphogenetic program in polarized Madin-Darby canine kidney cells resembling an epithelial-to-mesenchymal transition. We propose that altered cell-cell and cell matrix interactions may serve as an early event in H. pylori-induced carcinogenesis.

Abstract

This article focuses on the five most common bacterial enteropathogens of the developed world--Helicobacter pylori, Escherichia coli, Shigella, Salmonella, and Campylobacter--from the perspective of how they cause disease and how they relate to each other. Basic and recurring themes of bacterial pathogenesis, including mechanisms of entry, methods of adherence, sites of cellular injury, role of toxins, and how pathogens acquire particular virulence traits (and antimicrobial resistance), are discussed.

Abstract

The epithelial apical-junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical-junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical-junctional complex of the Ig superfamily--junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor--are important regulators of junction structure and function and represent critical targets of microbial virulence gene products.

Abstract

Helicobacter pylori translocates the protein CagA into gastric epithelial cells and has been linked to peptic ulcer disease and gastric carcinoma. We show that injected CagA associates with the epithelial tight-junction scaffolding protein ZO-1 and the transmembrane protein junctional adhesion molecule, causing an ectopic assembly of tight-junction components at sites of bacterial attachment, and altering the composition and function of the apical-junctional complex. Long-term CagA delivery to polarized epithelia caused a disruption of the epithelial barrier function and dysplastic alterations in epithelial cell morphology. CagA appears to target H. pylori to host cell intercellular junctions and to disrupt junction-mediated functions.

Abstract

Although intracellular Helicobacter pylori have been described in biopsy specimens and in cultured epithelial cells, the fate of these bacteria is unknown. Using differential interference contrast (DIC) video and immunofluorescence microscopy, we document that a proportion of cell-associated H. pylori enter large cytoplasmic vacuoles, where they remain viable and motile and can survive lethal concentrations of extracellular gentamicin. Entry into vacuoles occurs in multiple epithelial cell lines including AGS gastric adenocarcinoma, Caco-2 colon adenocarcinoma and MDCK kidney cell line, and depends on the actin cytoskeleton. Time-lapse microscopy over several hours was used to follow the movement of live H. pylori within vacuoles of a single cell. Pulsed, extracellular gentamicin treatments show that the half-life of intravacuolar bacteria is on the order of 24 h. Viable H. pylori repopulate the extracellular environment in parallel with the disappearance of intravacuolar bacteria, suggesting release from the intravacuolar niche. Using electron microscopy and live fluorescent staining with endosomal dyes, we observe that H. pylori-containing vacuoles are similar in morphology to late endosomal multivesicular bodies. VacA is not required for these events, as isogenic vacA- mutants still enter and survive within the intravacuolar niche. The exploitation of an intravacuolar niche is a new aspect of the biological life cycle of H. pylori that could explain the difficulties in eradicating this infection.

Abstract

Medical students often struggle to appreciate the clinical relevance of material taught in the preclinical years. The authors believe videos could be effectively used to interweave a patient's illness script with foundational basic science concepts.In collaboration with four other U.S. medical schools, educators at the Stanford University School of Medicine created 36 short, animated, patient-centered springboard videos (third-person, narrated accounts of authentic patient cases conveying foundational pathophysiology) in 2014. The videos were used to introduce students to 36 content modules, created as part of a microbiology, immunology, and infectious diseases curriculum. The videos were created with input from faculty content experts and in some cases medical students, and were piloted using a flipped classroom pedagogical approach in January 2015-June 2016.Student feedback from course evaluations and focus groups was analyzed using a mixed-methods approach. On the course evaluations, the majority of students rated the patient-centered videos positively, and the majority of comments on the videos were positive, highlighting both enhanced engagement and enhanced learning and retention. Comments from focus groups mirrored the course evaluation comments and highlighted different usage patterns for the videos.The authors will continue to gather and analyze data from schools using the videos as part of their core preclinical curriculum, and will produce similar videos for use in other areas of undergraduate medical education. These videos could support students' review of content taught previously and be repurposed for use in continuing and graduate medical education, as well as patient education.

Abstract

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth.

Abstract

Genomic technologies have significantly advanced our understanding of the composition and diversity of host-associated microbial populations. However, their spatial organization and functional interactions relative to the host have been more challenging to study. Here we present a pipeline for the assessment of intestinal microbiota localization within immunofluorescence images of fixed gut cross-sections that includes a flexible software package, BacSpace, for high-throughput quantification of microbial organization. Applying this pipeline to gnotobiotic and human microbiota-colonized mice, we demonstrate that elimination of microbiota-accessible carbohydrates (MACs) from the diet results in thinner mucus in the distal colon, increased proximity of microbes to the epithelium, and heightened expression of the inflammatory marker REG3β. Measurements of microbe-microbe proximity reveal that a MAC-deficient diet alters monophyletic spatial clustering. Furthermore, we quantify the invasion of Helicobacter pylori into the glands of the mouse stomach relative to host mitotic progenitor cells, illustrating the generalizability of this approach.

Abstract

Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non-phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZHP , localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZHP localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZHP for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZHP determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZHP and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities.

Abstract

We report the second pediatric case of New Delhi metallo-beta-lactamase (NDM-1)-producing Enterobacteriaceae in the United States in a girl from India who presented to a teaching hospital in Northern California with cystitis due to NDM-1-producing E. coli and K. pneumoniae. Laboratory methods included various phenotypic antimicrobial susceptibility testing assays, as well as PCR assays for carbapenemase-encoding genes. Laboratory challenges included a false negative modified Hodge test and reversion of carbapenem resistance in the E. coli strain. The limited number of effective antimicrobial agents and the lack of pediatric-specific safety and efficacy data for these drugs presented significant therapeutic challenges.

Abstract

Chronic infection with the bacterial pathogen Helicobacter pylori causes gastric disorders, ranging from chronic gastritis to gastric adenocarcinoma. Only a subset of infected persons will develop overt disease; most remains asymptomatic despite lifelong colonization. This study aims to elucidate the differential susceptibility to H pylori that is found both across and within populations.We have established a C57BL/6 mouse model of H pylori infection with a strain that is capable of delivering the virulence factor cytotoxin-associated gene A (CagA) into host cells through the activity of a Cag-pathogenicity island-encoded type IV secretion system.Mice infected at 5-6 weeks of age with CagA(+)H pylori rapidly develop gastritis, gastric atrophy, epithelial hyperplasia, and metaplasia in a type IV secretion system-dependent manner. In contrast, mice infected during the neonatal period with the same strain are protected from preneoplastic lesions. Their protection results from the development of H pylori-specific peripheral immunologic tolerance, which requires transforming growth factor-β signaling and is mediated by long-lived, inducible regulatory T cells, and which controls the local CD4(+) T-cell responses that trigger premalignant transformation. Tolerance to H pylori develops in the neonatal period because of a biased ratio of T-regulatory to T-effector cells and is favored by prolonged low-dose exposure to antigen.Using a novel CagA(+)H pylori infection model, we report here that the development of tolerance to H pylori protects from gastric cancer precursor lesions. The age at initial infection may thus account for the differential susceptibility of infected persons to H pylori-associated disease manifestations.

Abstract

Helicobacter pylori is a gram-negative pathogen that colonizes the stomachs of over half the world's population and causes a spectrum of gastric diseases including gastritis, ulcers, and gastric carcinoma. The H. pylori species exhibits unusually high levels of genetic variation between strains. Here we announce the complete genome sequence of H. pylori strain G27, which has been used extensively in H. pylori research.

Abstract

The association of Helicobacter pylori with gastric cancer is the best-studied relationship between a bacterial infection and cancer. Other bacterial pathogens in humans and rodents are now being recognized as potentially having a direct role in carcinogenesis. Thus, it might be possible to understand the pathogenesis and prevention of certain cancers by studying the bacterial infections associated with them, and their effects on the host. However, the mechanisms by which bacteria contribute to cancer formation are complex, and recent investigations show that they involve the interplay between chronic inflammation, direct microbial effects on host cell physiology and, ultimately, changes in tissue stem cell homeostasis.

Helicobacter pylori and gastric cancer: What can be learned by studying the response of gastric epithelial cells to the infection?AACR Special Conference on Colorectal Cancer - Molecular Pathways and TherapiesMueller, A., FALKOW, S., Amieva, M. R.AMER ASSOC CANCER RESEARCH.2005: 1859–64

Abstract

The development of gastric adenocarcinoma is closely linked to chronic infection with the bacterial pathogen Helicobacter pylori. One Helicobacter-specific virulence factor in particular, the CagA protein, has emerged as a main effector molecule in the interaction of H. pylori with gastric epithelial cells and has been implicated in gastric carcinogenesis. This review highlights the latest insights that have been gained into the pathogenesis of the disease by transcriptional profiling approaches studying gene expression in normal gastric tissue and gastric cancer tissue from human biopsy material as well as animal models of Helicobacter infection. The potential role of CagA as a bacterial oncoprotein is also discussed.

Abstract

A 14-year-old girl was seen at a community clinic with a chief complaint of abdominal pain and fevers and was treated with oral ciprofloxacin for presumed pyelonephritis. She became tachycardic and hypotensive after her first dose of antibiotic, and she developed disseminated intravascular coagulation. She was admitted to our hospital for presumed sepsis. Her outpatient peripheral blood smear was reviewed, revealing spirochetes consistent with Borrelia sp. To our knowledge this is the first reported case of the Jarisch-Herxheimer reaction to ciprofloxacin.

Imaging of dynamic changes of the actin cytoskeleton in microextensions of live NIH3T3 cells with a GFP fusion of the F-actin binding domain of moesin.BMC cell biologyLitman, P., Amieva, M. R., FURTHMAYR, H.2000; 1: 1-?

Abstract

The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin.C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent.Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex.

Abstract

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

Abstract

Ezrin belongs to a family of plasma membrane-cytoskeleton linking, actin binding proteins (Ezrin-radixin-Moesin family) involved in signal transduction, growth control, cell-cell adhesion, and microvilli formation.The expression of ezrin was examined in glomerular cells in culture, during kidney development, in the mature kidney, and in five different experimental kidney disease models in the rat.Ezrin was specifically expressed in glomerular epithelial cells in developing glomeruli in mature glomeruli and in glomerular epithelial cells in culture. Distinct from its other family members, moesin and radixin, which are predominantly expressed in glomerular endothelial and mesangial areas, ezrin protein (by immunohistochemistry) was specifically and exclusively modulated during podocyte injury and regeneration. Ezrin immunohistochemistry was able to visualize cell body attenuation, pseudocysts, and in particular vacuolation of injured podocytes, a feature that usually has to be identified at the ultrastructural level, and was strikingly increased in binucleated podocytes or podocytes that were partially or completely detached from the underlying GBM (frequently also binucleated). Infiltrating macrophages also express ezrin, but can easily be differentiated from podocytes by their round shape and higher level of expression.Ezrin likely has a role in the cytoskeletal organization, such as reassembling of acting filaments accompanying podocyte injury and regeneration. Since suitable light microscopic markers for the identification of glomerular epithelial cells are rare, ezrin may also be a useful marker for podocytes in normal and injured glomeruli.

Abstract

Re-epithelialization of skin wounds depends upon the migration of keratinocytes from the cut margins of the wound and is enhanced when human keratinocytes are covered with occlusive dressings that induce hypoxia. In this study, two independent migration assays were used to compare cellular motility on connective tissue components under normoxic or hypoxic conditions. Human keratinocytes apposed to collagens or fibronectin exhibited increased motility when subjected to hypoxic (0.2 or 2% oxygen) conditions compared with normoxic (9 or 20% oxygen) conditions. When compared with normoxic cells, hypoxic keratinocytes exhibited increased expression and redistribution of the lamellipodia-associated proteins (ezrin, radixin, and moesin). Furthermore, hypoxic keratinocytes demonstrated decreased secretion of laminin-5, a laminin isoform known to inhibit keratinocyte motility. Hypoxia did not alter the number of integrin receptors on the cell surface, but did induce enhanced secretion of the 92-kD type IV collagenase. These data demonstrate that hypoxia promotes human keratinocyte motility on connective tissue. Hypoxia-driven motility is associated with increased expression of lamellipodia proteins, increased expression of collagenase and decreased expression of laminin-5, the locomotion brake for keratinocytes.

Abstract

To determine, whether 558Thr in the carboxyl-terminal domain of moesin is phosphorylated in cells other than platelets, rabbit phosphorylation state-specific antibodies were made to the chemically phosphorylated synthetic hexapeptide KYKpTLR of the moesin sequence, as well as to the unphosphorylated form. The affinity-purified antibody populations were specific for either the phosphorylated or the unmodified peptide conjugated to BSA. Site-specific phosphorylation of moesin is detected in RAW macrophages by Western blot analysis, and immunofluorescence studies demonstrate that phosphorylated moesin is localized in filopodial protrusions. After pretreatment with the phosphatase inhibitor calyculin A, a similar effect to that seen in platelets in found, namely a substantial increase in moesin phosphorylation at 558Thr and redistribution of phospho-moesin together with F-actin into one or more ring-like structures in the cytoplasm, presumably due to binding of phosphorylated moesin to F-actin.

Abstract

The expression of the two cytoskeletal linking proteins, moesin and radixin, was examined in experimental mesangial proliferative nephritis in rats (anti-Thy1 model). Moesin and radixin mRNA and protein are constitutively expressed in all cell types of normal rat glomeruli, except podocytes. In the anti-Thy1 model the expression of moesin and radixin was increased in infiltrating macrophages and in activated, alpha-smooth muscle actin-positive mesangial cells and was concentrated in the cellular extensions of mesangial cells in areas of glomerular remodelling. Studies using neutralizing antibodies demonstrated that the increase in moesin and radixin expression by mesangial cells is mediated by PDGF, but not bFGF. The increase in these cytoskeletal proteins appears to be regulated primarily (radixin) or partially (moesin) posttranscriptionally. The data suggest that PDGF mediated upregulation of the cytoskeletal proteins, moesin and radixin, is important for cell migration and other changes that accompany the coordinated restoration of glomerular architecture after injury.

Abstract

The phosphorylation and localization of the membrane-linking protein moesin was analyzed during early activation of platelets with thrombin. Activated platelets elaborate filopodia and spread to assume flat pancake-like shapes, and moesin is localized in filopodia and cell body. In resting platelets, approximately 25% of moesin molecules are phosphorylated as shown by metabolic labeling with 32P(i) and by isoelectric focusing. Within seconds after exposure to thrombin, phosphorylation increases, reaching a maximum of 35% labeled molecules by 1 min, followed by a decrease to a new basal level within 5 min. This modification affects a single residue, Thr558, which is located within or close to a binding site for F-actin. Rapid shifts (0-100%) in the number of phosphorylated molecules are observed in the presence of inhibitors of serine/threonine kinases and phosphatases. Inhibitors affecting tyrosine phosphorylation also modulate phosphorylation at this site suggesting that the enzymes involved in the modification of Thr558 are regulated by tyrosine phosphorylation. Platelets respond to both extremes of modification by forming extremely long filopodia and the absence of spreading on glass. Completely phosphorylated moesin is concentrated together with F-actin in the center of the cell. The rapid modification of moesin at or near its actin-binding domain suggests a model for regulated membrane-cytoskeleton interaction during cell activation.

Abstract

Moesin, a member of the talin-4.1 superfamily, is a linking protein of the submembraneous cytoskeleton. It is expressed in variable amounts in cells of different phenotypes such as macrophages, lymphocytes, fibroblastic, endothelial, epithelial, and neuronal cell lines. In this report we show that moesin is not randomly distributed throughout the cortical cytoskeleton, but rather that it is concentrated in specialized microdomains. It is localized in the intracellular core of microextensions known as filopodia, microvilli, microspikes, and retraction fibers. This subcellular distribution follows closely the dynamic changes in cell shape that take place when cells attach, spread, and move spontaneously or in response to extracellular signals. This suggests a similar function for moesin in diverse cell types related to the dynamic restructuring of domains of the plasma membrane and underlying membrane skeleton. Support for this comes from studies on PC-12 cells, which respond to NGF by extending neurites and moesin is redistributed from a diffuse localization to growth cone filopodia. In fibroblastic (NIH3T3) or macrophage (RAW264.7) cell lines, moesin is found in filopodia appearing at random on the cell surface soon after the cells are placed in culture, begin to attach, and spread. In polarized epithelial cells (LLC-PK1), moesin is associated with peripheral filopodia and apical microvilli. The cellular microextensions containing moesin are devoid of microtubules, focal contact proteins such as vinculin, and cortical cytoskeletal elements such as protein 4.1, but they do contain varying amounts of actin microfilaments. This localization of moesin in microextensions is not influenced by cytochalasin B. Treatment of cells with phorbolester (PMA) causes rapid cell spreading, disappearance of filopodia and retraction fibers, and moesin does not accumulate in the actin-rich lamellae that form at the cellular edges. After removal of PMA, cells retract and moesin again becomes concentrated in filopodia and retraction fibers. These studies support the hypothesis that filopodia, retraction fibers, and other microextensions of the plasma membrane are unique cellular microdomains with characteristic submembraneous components. Moesin could be involved in the dynamic restructuring of such microdomains by regulating binding interactions between the plasma membrane and the actin cytoskeleton.

Abstract

Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein.

Abstract

The early neurogenesis of the mouse olfactory nerve, from its exist at the nasal epithelium to its entrance into the embryonic telencephalon, has been investigated by using the rapid Golgi method and electron microscopy. Previously unrecognized anatomical and possible functional interrelationships between developing olfactory nerve axons and their sheath cells have been observed: 1) at their exit from sensory epithelium (nasal compartment), 2) at their contact with the CNS surface (intracranial compartment), and 3) at their entrance into the embryonic telencephalon (central nervous tissue compartment). Based on these observations the anatomy of the mouse olfactory nerve is herein redefined. Exiting olfactory nerve axons and sheath cells from the same regions of the nasal epithelium establish an early association which is maintained up to their terminal glomerular neuropile. No disruptions have been found in either the olfactory nerve axons or in the continuity of their sheath cells from exit at the nasal epithelium to entrance into the developing olfactory bulb. Corresponding olfactory nerve axons with their sheath cells enter together and become incorporated into the developing olfactory bulb as units. Consequently, the cellular envelope of the olfactory glomerulus must be composed of olfactory sheath cells rather than of glial (astroglial) cells from the CNS. With this simple anatomical arrangement, a topographic map of the sensory epithelium could be established progressively in the developing olfactory bulb. Eventually, "regenerating" olfactory nerve axons from different nasal regions could be guided by their specific sheath cell conduits toward their target glomeruli; hence, the olfactory message may be maintained undisturbed throughout the life span of the animal. In addition, olfactory nerve axons establish synaptic-like contacts with their corresponding sheath cells prior to or during the perforation of the CNS surface. Reciprocal recognition between corresponding axons and their sheath cells at this crucial stage in their neurogenesis may play a significant role in the establishment of their terminal glomerulus. This new concept of the anatomy of the mammalian olfactory nerve should provide insights helpful in clarifying some of the still-unresolved questions regarding the structural and functional organizations of this primitive system.